Cytotoxic necrotizing factor type 1 (CNF1) is a member of a family of bacterial toxins that deamidate single glutamine residues in RhoA, Rac, and Cdc42 and thereby constitutively activate these small GTPases. These deamidation events trigger a myriad of effects on the target cells such as actin cytoskeleton rearrangements, cell cycle abnormalities, and alterations in signaling pathways. CNF1 and a membrane-lytic toxin, hemolysin (Hly), are often coexpressed by Escherichia coli strains that cause urinary tract infections (UTIs), i.e., cystitis or pyelonephritis, or acute prostatitis. In fact, the cnf1 locus and a hly operon are co-transcribed and co-regulated from a prototypic uropathogenic E. coli (UPEC) strain during culture in vitro. Of particular relevance to this proposal, CNF1/Hly-producing UPEC isolates are more frequently isolated from humans with blood and high levels of certain pro-inflammatory cytokines in their urine than are toxin-negative UPEC. The latter observation is consistent with our prior findings that CNF1+ UPEC strains elicit a more intense inflammatory response than do isogenic CNF1- mutants in a mouse model of ascending UTI and in a rat model of acute prostatitis and that the CNF1-positive UPEC strain CP9 survives better than does its cnf1 isogenic mutant in human and mouse polymorphonuclear leukocytes (PMNs). We also reported that Hly provokes loss of surface uroepithelial cells in culture and a 3-D organoid model, and we recently found that Hly damages the uroepithelium and evokes hemorrhage in the bladders of mice 24 hours after intraurethral inoculation with CP9. We therefore theorize that CNF1 and Hly enhance the pathogenicity of UPEC strains by: I.) promoting uroepithelial cell shedding and tissue hemorrhage (Hly);ii.) evoking a large influx of potentially tissue-damaging PMNs (CNF1 and Hly) while simultaneously protecting the bacterium from phagocyte-mediated killing (CNF1), and;iii.) eliciting submucosal edema (CNF1). The specific aims designed to test this hypothesis are to: 1.) delineate the impact of CNF1 and Hly alone and together on the levels of blood, PMNs, and selected pro-inflammatory cytokines in the urine of mice during the first 24 hours after intraurethral infection with CP9 and its cnf1 and hlyA1 single and double mutants and to more broadly compare the host response to these isogenic strains through microarray transcriptional analyses of infected bladders;2.) monitor expression kinetics of cnf1 and the contiguous hly operon by real time RT-PCR in the urine and/or bladders of CP9-challenged mice to ask whether cnf1 and the linked hly operon are co-transcribed in vivo;3.) determine whether CNF1 actually modifies small GTPases in vivo by measuring the extent of activation of Rho, Rac and Cdc42 in uroepithelial cells from bladders of mice infected with CP9 or its CNF mutant;and, 4.) attempt to reduce the extent of inflammation and damage evoked by CP9 through parenteral and/or mucosal immunization of mice with a CNF1/Hly toxoid cocktail. PUBLIC HEALTH RELEVANCE: UTIs are among the most common bacterial diseases of adults, with women affected disproportionately to men. This project will lead to a better understanding of the relative importance of CNF1 and Hly in UPEC-mediated disease and may pave the way for the design of novel preventative and therapeutic strategies against UTIs.